Device and method for concentrating and detecting pathogenic microbes from blood products and/or their derivatives

ABSTRACT

A method for detecting contaminating microbes possibly present in a blood product including blood cells including a) subjecting a sample of the blood product to an aggregation treatment of the blood cells, b) substantially eliminating aggregates formed in step (a) by passage of the sample over a first filter allowing passage of contaminating microbes, but not cell aggregates, c) selectively lysing residual cells of the filtrate obtained in step (b), d) recovering the contaminating microbes by passage of the lysate from step (c) over a second filter allowing passage of cellular debris, e) adding a marker agent of the contaminating microbes either during step (a) or step (c), and f) analyzing material on the second filter to detect labeled contaminating microbes possibly retained by the second filter; and a device for concentrating contaminating microbes possibly present in a blood product including blood cells including a first watertight, sterile tank containing at least one blood cell aggregation agent and, optionally, at least one agent for labeling pathogenic microbes, a second watertight, sterile tank containing at least one lysis agent for blood cells and, optionally, at least one agent for labeling pathogenic microbes, a first filter located between the first and second tanks and capable of retaining aggregates formed in said first tank, a second filter located downstream of the second tank and capable of retaining possible contaminating pathogenic microbes, and a watertight, sterile connector placed between the first tank and the first filter, between the first filter and the second tank, and between the second tank and the second filter.

RELATED APPLICATION

[0001] This is a continuation of International Application No.PCT/FR02/03132, with an international filing date of Sep. 13, 2002 (WO03/025207, published Mar. 27, 2003), which is based on French PatentApplication No. 01/11873, filed Sep. 13, 2002.

FIELD OF THE INVENTION

[0002] This invention relates a method for concentrating pathogenicmicrobes possibly present in blood products or their derivatives as wellas detecting the microbes thereby concentrated to monitor thepathogenicity of the blood products.

BACKGROUND

[0003] The term “blood product” is understood to mean whole blood aswell as any preparation stemming from the fractionation of whole blood,optionally comprising cellular components. The following can be cited asexamples of blood products: concentrates of red cells or platelets, butalso plasma or serum preparations.

[0004] Detection of contaminations of blood products and theirderivatives by different pathogenic microbes such as bacteria, viruses,molds, yeasts and others, is one of the major problems facing the publichealth authorities at present as well as the blood transfusionindustries. Detection tests exist, but they cannot be used on a routinebasis at present. The principal problems presented by most of the testsfor detecting such pathogenic microbes among a population orsubpopulation of blood cells are that most of the treatments that aresupposed to selectively extract the pathogenic microbes simultaneouslycause an elimination of these microbes. This elimination leads almostsystematically to an underassessment of the presence of the microbes inthe blood product tested and, thus, to an increase in the health carerisk. It would therefore be advantageous to provide a new, rapid,sensitive method for detecting contamination of a blood product or itsderivative by pathogenic microbes.

SUMMARY OF THE INVENTION

[0005] This invention relates to a method for detecting contaminatingmicrobes possibly present in a blood product including blood cellsincluding a) subjecting a sample of the blood product to an aggregationtreatment of the blood cells, b) substantially eliminating aggregatesformed in step (a) by passage of the sample over a first filter allowingpassage of contaminating microbes, but not cell aggregates, c)selectively lysing residual cells of the filtrate obtained in step (b),d) recovering the contaminating microbes by passage of the lysate fromstep (c) over a second filter allowing passage of cellular debris, e)adding a marker agent of the contaminating microbes either during step(a) or step (c), and f) analyzing material on the second filter todetect labeled contaminating microbes possibly retained by the secondfilter.

[0006] This invention also relates to a device for concentratingcontaminating microbes possibly present in a blood product includingblood cells including a first watertight, sterile tank containing atleast one blood cell aggregation agent and, optionally, at least oneagent for labeling pathogenic microbes, a second watertight, steriletank containing at least one lysis agent for blood cells and,optionally, at least one agent for labeling pathogenic microbes, a firstfilter located between the first and second tanks and capable ofretaining aggregates formed in the first tank, a second filter locateddownstream of the second tank and capable of retaining possiblecontaminating pathogenic microbes, and watertight, sterile connectorsplaced between the first tank and the first filter, between the firstfilter and the second tank, and between the second tank and the secondfilter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Other advantages and characteristics of the invention will becomeapparent from the examples below and the attached figures in which:

[0008]FIG. 1 is a graph illustrating platelet counting after havingbrought platelet concentrates into contact with different concentrationsof thrombin;

[0009]FIG. 2 is a graph illustrating platelet counting after aggregationin the presence of ADP for two platelet samples;

[0010]FIG. 3 is a graph showing the platelet counting in the presence ofCD9 antibodies (clone SN4) for four platelet samples;

[0011]FIG. 4 is a graph showing the results of platelet counting in thepresence of increasing doses of antibody CD9 (clone 6B1) used in 3 mL ofplatelet concentrate;

[0012]FIG. 5 is a graph showing a dose response curve obtained in thepresence of increasing concentrations of antibody CD9 (clone SN4);

[0013]FIG. 6 is a graph illustrating the agglutination of red cells inthe presence of Phaseolus vulgaris lectin used at a concentration of 200μg/ml for three samples of red cell concentrates;

[0014]FIG. 7 is a graph showing the effects of the lysis solution on therecovery of different pathogenic microbes in pure cultures;

[0015]FIG. 8 is a graph showing the effect of the lysis solution on theplatelet number of two platelet samples;

[0016]FIG. 9 is a graph illustrating the effects of lysis on theerythrocyte count of two different samples of red cell concentrate;

[0017]FIG. 10 is a graph illustrating the counting of Escherichia colibacteria within a platelet sample using polyethylene imine (PEI) as apermeabilization agent to increase penetration of the marker;

[0018]FIG. 11 is a graph illustrating the effect of the concentration ofN-octyl β-D-glucopyranoside in the lysis solution on the pure bacteriacultures; and

[0019]FIG. 12 is a schematic perspective view illustrating a preferredembodiment of the device for concentrating pathogenic microbes accordingto aspects of the invention.

DETAILED DESCRIPTION

[0020] The method according to aspects of the invention is remarkable inthat it is performed directly on a sample stemming from a blood productcollected from a subject without prior treatment or dilution. The methodfor detecting pathogenic microbes comprises selectively concentratingthe pathogenic microbes, then, once they have been concentrated,detecting them by techniques known in the art. Selective concentrationof the pathogenic microbes is performed by sequential or simultaneouselimination of the different populations of blood cells present in ablood product sample.

[0021] The method for concentrating pathogenic microbes according toaspects of the invention comprises a first step of concentrating thepathogenic microbes consisting of reducing the blood cell populations byselective aggregation of the cells, followed by a filtration step tocollect in the filtrate the unaggregated, concentrated pathogenicmicrobes and retain on the filter the blood cell aggregates.

[0022] The term “aggregation”, in the context of this invention, isunderstood to mean any action leading to the formation of cellaggregates. The term “cell aggregates” is understood to mean any groupof cells comprising more than two cells and the size of which is greaterthan that of an isolated cell. In the context of this invention, anaggregate can be obtained either by aggregation, such as aggregation ofplatelets subsequent to their activation, an agglutination, such asagglutination of red cells obtained when they are in the presence ofparticular molecules, or bringing together cells induced by a change inthe electrostatic charge of their membranes or other adhesion mechanismsor bringing together cells leading to the grouping together of more thantwo cells.

[0023] According to preferred embodiments, the aggregation of differentpopulations of blood cells can be performed by compounds inducingplatelet aggregation or compounds inducing specific agglutination of redcells. It is known, for example, that in the presence of certaincompounds, the platelets have the capacity to aggregate with each other.These aggregates can be easily separated from the pathogenic microbes byfiltration. The red cells also have several agglutination properties.

[0024] The method for concentrating pathogenic microbes according toaspects of the invention optionally comprises a second step of reducingthe concentration of populations of the predominant cells in the blood,i.e., the platelets and the red cells, consisting of lysing theunaggregated cells isolated in the first aggregation step.

[0025] This second step of reducing the concentration of blood cellpopulations enables a reduction on the order of 4 log (from about 10⁹ toabout 10⁵ cells/ml) regarding the concentration of platelets and on theorder of 5 log (from about 10¹⁰ to about 10⁵) regarding theconcentration of red cells.

[0026] More precisely, this invention provides a method forconcentrating contaminating microbes possibly present in a blood productcomprising blood cells, comprising the following steps:

[0027] a) a sample of the blood product is subjected to an aggregationtreatment of the blood cells,

[0028] b) aggregates formed in step (a) are eliminated by passage of thetreated sample over a first filter allowing passage of the contaminatingmicrobes, but not the cell aggregates,

[0029] c) residual cells of the filtrate obtained in step (b) are lysedselectively,

[0030] d) contaminating microbes are recovered by passage of the lysatefrom step (c) over a second filter allowing passage of the cellulardebris.

[0031] According to a preferred embodiment, the method comprises asupplementary step of analysis of the second filter to detect thecontaminating microbes possibly retained on it. The methodadvantageously comprises the addition of a marker agent of thecontaminating microbes either during the aggregation of step (a), orduring the lysis of step (c), or directly on the second filter duringthe analysis step (e).

[0032] A marker solution comprising an esterase substrate such asChemChrome V6 is an example of a marker agent of the pathogenic microbesdetectable by the method of this invention. Thus, it is possible to usea marker solution comprising a labeled antibody or a marker of nucleicacids. The marker is preferably fluorescent or coupled to a fluorochromeor an enzyme enabling degradation of a substrate thereby madefluorescent, with the possibility that the fluorescence can be detectedby an excitation laser.

[0033] The method also comprises addition of a permeabilization agent ofthe contaminating microbes which can be added to at least one of thesteps, either during the aggregation of step (a), or during the lysis ofstep (c), or directly on the second filter during the analysis of step(e), or during several of these steps.

[0034] Examples of permeabilization agents of the contaminating microbesinclude, but are not limited to, polyethylene imine, chlorhexidinediacetate, chlorhexidine digluconate, ethylene diamine tetraacetate acid(EDTA) alone or in combination with nisin as well as detergents such asN-octyl β-D-glucopyranoside, SDS, Tween, triton, Brij and the like.

[0035] According to a preferred embodiment, the blood cells of the bloodproduct are platelets or red cells or a mixture of these two. Accordingto another preferred embodiment, the blood cells of the blood productare platelets and the aggregation treatment of step (a) comprisesbringing the sample into contact with an aggregation compositioncomprising at least one of the aggregation agents selected from thegroup comprising: 1) a specific antibody of a platelet antigen, 2) astrong agonist of platelet activation selected from among: thrombin,TRAP (thrombin receptor activating peptide), trypsin, collagen,thromboxane A2 or ionophore A23187, and 3) a weak agonist of plateletaggregation selected from among ADP, adrenalin, arachidonic acid, VonWillebrand factor, serotonin or epinephrine.

[0036] The concentration of CD9 antibody specific of a platelet antigenin the aggregation composition is advantageously between about 0.5 μg/mland about 100 μg/ml, preferably between about 5 μg/ml and about 40μg/ml.

[0037] The concentration of strong agonist in the aggregationcomposition is advantageously between:

[0038] about 0.5 IU/ml and about 100 IU/ml, preferably between about 1IU/ml and about 20 IU/ml, for a thrombin type agonist;

[0039] about 5 μM and about 200 μM, preferably between about 10 andabout 100 μM, for a TRAP type agonist;

[0040] about 1 nM and about 500 nM, preferably between about 10 nM andabout 300 nM, for a trypsin type agonist;

[0041] about 0.05 μg/ml and about 50 μg/ml, preferably between about 1μg/ml and about 20 μg/ml, for a collagen type agonist;

[0042] about 0.01 μg/ml and about 5 μg/ml, preferably between about 0.1and about 1 μg/ml, for a thromboxane A2 type agonist;

[0043] about 0.005 mg/ml and about 1 mg/ml, preferably between about0.05 and about 0.5 mg/ml, for a PAF type agonist;

[0044] about 0.1 μM and about 100 μM, preferably between about 1 μM andabout 20 μM, for an ionophore A23187 type agonist.

[0045] The concentration of weak agonist in the aggregation compositionis advantageously between:

[0046] about 0.5 μM and about 100 μM, preferably between about 1 μM andabout 20 μM, for an agonist of the ADP, adrenalin or epinephrine type;

[0047] about 0.001 mM and about 10 mM, preferably between about 0.01 mMand about 5 mM, for an agonist of the arachidonic acid type;

[0048] about 0.001 mg/ml and about 1 mg/ml, preferably between about0.01 mg/ml and about 0.5 mg/ml, for an agonist of the Von Willebrandfactor type;

[0049] about 0.05 μand about 100 μM, preferably between about 0.01 μMand about 50 μM, for an agonist of the serotonin type.

[0050] The specific antibody of a platelet antigen is preferablyselected from among: an anti-CD, CD32, anti-PTA1, CD42, anti-GpIIb/IIIaand anti-GpIV antibody.

[0051] According to another embodiment, the blood product comprises redcells and the aggregation treatment of step (a) comprises bringing thesample into contact with an agglutination composition comprising atleast one agglutination agent selected from among the lectins,polyethylene imine, polyvinylpyrrolidone (PVP), gelatins, dextrans orpolyethylene glycols (PEG). The lectins advantageously have anerythroagglutinin activity. Most preferably, the lectins are selectedfrom among the lectins of Phaseolus vulgaris, Vicia sativa, Vicia fabaor Erythrina corallodendron, Lens culinaris, Phytolacca Americana orTriticum vulgaris. The concentration of Phaseolus vulgaris type lectinin the agglutination composition is advantageously between about 10μg/ml and about 200 μg/ml.

[0052] The concentration of polyethylene imine in the agglutinationcomposition is advan-tageously between about 0.1% (weight/volume) andabout 40% (weight/volume). The dextrans are most preferably selectedfrom among Dextran 70, Dextran 100, Dextran 500 and the like. Theconcentration of dextran in the agglutination composition isadvantageously between about 0.1% (weight/volume) and about 40%(weight/volume).

[0053] The PEG compounds are most preferably selected from among PEG35,PEG and the like. The concentration of PEG in the agglutinationcomposition is advantageously between about 0.05% (weight/volume) andabout 40% (weight/volume). The concentration of gelatin in theagglutination composition is advantageously between about 0.5%(weight/volume) and about 40% (weight/volume).

[0054] The PVP compounds are most preferentially selected from amongPVP-40, PVP-360 and the like. The concentration of PVP in theagglutination composition is advantageously between about 0.05%(weight/volume) and about 40% (weight/volume).

[0055] Lysis of the cells of step (c) is advantageously performed with alysis solution comprising one or more detergents selected from saponin,SDS, Tween 20, Triton X 100, Brij 96, Polido-canol, N-octylβ-D-glucopyranoside and sodium carbonate. The lysis solution ispreferably constituted of a mixture of saponin, Triton X100 and Tween20. Most preferably, the lysis solution comprises saponin at aconcentration (expressed in weight/volume %) between about 0.005% andabout 0.5%, of Triton X100 at a concentration (expressed inweight/volume %) between about 0.001% and about 0.5% of Tween 20 at aconcentration between about 0.01% and about 1% and of N-octylβ-D-glucopyranoside at a concentration between about 0.1% and about0.5%.

[0056] Permeabilization of the bacteria is advantageously performed witha solution comprising one or more reagents selected from chlorhexidine(digluconate, diacetate), polyethylene imine, N-octylβ-D-glucopyranoside, nisin alone or in combination with EDTA. Thepermeabilization agents are preferably used in the case of chlorhexidineat a concentration (weight/volume) between about 0.0001% (weight/volume)and about 0.1% (weight volume), in the case of polyethylene imine at aconcentration between about 5 μg/ml and about 120 μg/ml, in the case ofN-octyl β-D-glucopyranoside at a concentration between about 0.1%(weight/volume) and about 0.5% (weight/volume) and in the case of nisinbetween about 0.1 μg/mL and about 0.5 μg/mL alone or in combination withEDTA at a concentration between about 1 mM and about 10 mM.

[0057] The method of the invention can be used to concentrate and detectnumerous contaminating microbes of blood products such as aerobic andanaerobic bacteria, molds, yeasts, live and/or dead bacterial spores.The size of the pores of the first filter is advantageously betweenabout 2 μm and about 20 μm. The size of the pores of the second filteris advantageously between about 0.2 μm and about 2 μm.

[0058] Detection of the contaminating microbes of step (e) of the methodof the invention is advantageously performed in an enclosed device. Mostpreferably, the contaminating microbes capable of being concentrated areselected from among the groups of aerobic and anaerobic bacteria, molds,yeasts and live and/or dead bacterial spores. The size of the pores ofthe first filter is between about 2 μm and about 20 μm, and the size ofthe pores of the second filter is between about 0.2 μm and about 2 μm.

[0059] This invention also provides a device for concentrating andlabeling contaminating microbes possibly present in a blood productcomprising, as shown in FIG. 12:

[0060] a first watertight, sterile tank (1) containing at least oneblood cell aggregation agent and possibly at least one agent forlabeling pathogenic microbes;

[0061] a second watertight, sterile tank (2) containing at least onelysis agent for blood cells and possibly at least one agent for labelingpathogenic microbes;

[0062] a first filter (3) placed between the first and second tanks andcapable of retaining the aggregates formed in the first tank;

[0063] a second filter (4) placed downstream of the second tank andcapable of retaining the possible contaminating pathogenic microbes; and

[0064] watertight, sterile connector (5) placed between the first tank(1) and the first filter (3), between the first filter (3) and thesecond tank (2), and between the second tank (2) and the second filter(4).

[0065] According to a preferred embodiment, the device comprises awatertight, sterile connector (6) to connect the bag containing theblood product to the first sterile tank (1). The watertight, sterileconnection (6) connecting the bag containing the blood product to thefirst sterile tank is advantageously equipped with a reverse lock valve(7).

[0066] According to another preferred embodiment, the device comprises asampling device to sample a determined volume of the blood productdirectly from a storage bag of the product into the first tank (1).

[0067] The first watertight, sterile tank (1) is advantageously fittedwith a sample suctioning system (8). The suctioning system is preferablya piston. According to another preferred embodiment, the second filter(4) is enclosed in a membrane support composed of two parts that can beseparated for removing the filter. The device i advantageously enclosedand sterile.

EXAMPLES

[0068] I. Concentration of pathogenic microbes by an aggregation step

[0069] I.1 Aggregation of platelets

Example 1

[0070] Aggregation with a Strong Agonist: Thrombin

[0071] Thrombin is a strong agonist of platelet aggregation. Thrombinsolutions (reference T8885 Sigma) were prepared at a concentration of100 IU/ml when used at the rate of 10 IU/test and in diluted solutionform (100 μl of thrombin mother solution with the addition of 900 μl ofPBS buffer) when used at the rate of 1 IU/test.

[0072] Platelet aggregation by the intermediary of thrombin comprised:

[0073] placing 160 μl of platelet concentrate in a tube to which wasadded 20 μl of PBS buffer and 20 μl of thrombin;

[0074] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0075] 800 μl of PBS buffer was added to the tubes;

[0076] the content of the tubes was filtered on a filter with a porosityof 11 μm;

[0077] dilutions were created in series {fraction (1/20)} to {fraction(1/10)} from the filtrates;

[0078] 100 ml of each dilution of the filtrate on a CB04 membrane werefiltered;

[0079] an esterase labeling was performed; and

[0080] the platelets possibly retained on the membrane were counted.

[0081] Table 1 below illustrates the results obtained and shows thenumber of platelet aggregates obtained in the presence of thrombin usedat two different concentrations. FIG. 1 graphically illustrates theseresults. TABLE 1 Control 1 IU thrombin 10 IU thrombin PC 6 1584 374 1535490 PC 6 2622 1358 44 2737 1399 30

Example 2

[0082] Platelet Aggregation with Thrombin in the Presence of PathogenicMicrobes

[0083] The experimental studies performed to evaluate the aggregation ofpathogenic microbes in the presence of thrombin comprised the followingsteps:

[0084] a cryobead of E. coli was introduced into a tube of 9 ml oftryptone soy broth and incubated at 37° C. for 18-24 hours;

[0085] 160 μl of platelet concentrate was added to 20 μl of the E. colisuspension and 20 μl of thrombin;

[0086] the tube was agitated manually for 5 minutes at ambienttemperature;

[0087] 800 μl of PBS buffer was added;

[0088] filtration was performed through a filter of 11 μm porosity;

[0089] dilutions in series were performed: {fraction (1/20)} and{fraction (1/10)} and {fraction (1/10)};

[0090] filtration was performed on 100 μl of sample on a CB04 membrane;

[0091] labeling with esterase was performed; and

[0092] detection was performed.

[0093] Table 2 below shows the aggregation of the platelet concentrateswith thrombin in the presence of E. coli. TABLE 2 Pathogenic microbePlatelet preparation Thrombin Counting results E. coli — — 1921 — — 1999E. coli PC 6 — 657 763 E. coli PC 6  1 IU 140 167 E. coli PC 6 10 IU 109122

[0094] A clot appeared almost instantly after addition of the thrombin.

Example 3

[0095] Platelet Aggregation in the Presence of a Weak Agonist: ADP

[0096] ADP (Sigma) was used at a concentration of 200 μM in distilledwater.

[0097] The experimental studies performed to evaluate plateletaggregation in the presence of ADP comprised the following steps:

[0098] 400 μl of platelet concentrate was introduced into a tube towhich was added 50 μl of PBS buffer and 50 μl of ADP;

[0099] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0100] 500 μl of PBS buffer was added;

[0101] dilutions were performed in series of {fraction (1/20)} and{fraction (1/10)} and {fraction (1/10)};

[0102]100 μl of sample was filtered on a CB04 membrane;

[0103] labeling with esterase was performed; and

[0104] detection was performed.

[0105] The results illustrated in FIG. 1 show that, in the presence ofADP, the concentration of platelets was reduced by about 50% to about90%.

Example 4

[0106] Platelet Aggregation with ADP in the Presence of PathogenicMicrobes

[0107] Platelet aggregation in the presence of ADP comprised thefollowing steps:

[0108] a cryobead of Staphylococcus epidermidis was introduced into atube of 9 ml of tryptone soy broth and incubated at 37° C. for 18-24hours;

[0109] onto 400 μl of platelet concentrate there was added 50 μl of thesuspension of Staph. epidermidis and 50 μl of ADP;

[0110] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0111] 500 μl of PBS buffer was added;

[0112] filtration was performed on a filter with a porosity of 5 μm;

[0113] dilutions were made in series of {fraction (1/20)} and {fraction(1/10)} and {fraction (1/10)};

[0114]100 μl of the sample was filtered on a CB04 membrane;

[0115] labeling with an esterase was performed; and

[0116] detection was performed.

[0117] The pathogenic microbes were seeded at a high concentration toaugment a possible trapping effect. ADP was added to the platelets andthe bacteria at a concentration of 10 μM. TABLE 3 Pathogenic PlateletAggregation Concentration microbe preparation agent Results bacteria/mlStaphylococcus PC 6 none 590 6.0E+07 epidermidis 605 Staphylococcus PC 610 μM 492 4.7E+07 epidermidis ADP 441

[0118] The results obtained, shown in Table 3 above, show that 74% ofbacteria are recovered after the aggregation step.

Example 5a.

[0119] Platelet Aggregation in the Presence of a CD9 Antibody

[0120] It is known that the CD9 antibody induces platelet activationand, consequently, their aggregation. Tests were performed with two CD9clones, clone SN4 (Ancel, ref. 156-020, con-centration 100 μg/ml) andclone 6B1 (Hemosystem).

[0121] The selective aggregation method implemented with these CD9antibodies comprised the following steps:

[0122] 400 μl of platelet concentrate was added to 50 μl of PBS bufferand 50 μl of CD9 antibody;

[0123] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0124] 500 μl of PBS buffer was added;

[0125] filtration was performed on a filter with a porosity of 5 μm;

[0126] dilutions were performed in series of {fraction (1/20)} and{fraction (1/10)} and {fraction (1/10)};

[0127]100 μl of sample was filtered on a CB04 membrane;

[0128] labeling was performed with an esterase; and

[0129] detection was performed.

[0130] The CD9 antibody (clone SN4) was used in the presence ofplatelets at a final concen-tration of 10 μg/ml. FIG. 2 illustratesplatelet aggregation obtained in the presence of antibody CD9 (cloneSN4). A dose-response curve was established to evaluate theconcentration of anti-body CD9 required for platelet aggregation.

[0131] Method:

[0132] 400 μl of platelet concentrate placed in tubes was added todifferent dilutions of antibody, with final concentrations ranging from0 to 20 μl/ml of antibody CD9;

[0133] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0134] 500 μl of PBS buffer was added;

[0135] filtration was performed on a filter with a porosity of 5 μm;

[0136] dilutions were performed in series: four {fraction (1/10)}dilutions;

[0137] 100 μl of sample was filtered on a CB04 membrane;

[0138] labeling was performed with an esterase; and

[0139] detection was performed.

[0140]FIGS. 3 and 5 show, respectively, the counting results foraggregated platelets in the presence of increasing doses of antibody CD9(clone SN4) and the dose-response curve thereby obtained. Counting ofthe residual platelets was performed with the analyzer.

[0141] The aggregation was dose dependent. The concentration of CD9 wasincreased as much as possible to increase the effect of aggregation.However, a compromise was established for detecting the bacteria. It wasdetermined that the platelet concentration could be reduced from 1 to 2log by using a concentration of about 10 μg/ml of antibody CD9.

Example 5b:

[0142] A dose-response curve was established to evaluate theconcentration of antibody CD9 (clone 6B1) for platelet aggregation.

[0143] Method:

[0144] 3 mL of platelet concentrate placed in tubes was added todifferent dilutions of antibody, with final concentrations ranging from2.5 μg/mL to 40 μl/mL of antibody CD9;

[0145] the tubes were agitated manually for 15 minutes at ambienttemperature;

[0146] filtration was performed on a filter with a porosity of 5 μm; and

[0147] platelet counting was performed as described above.

[0148] Attached FIG. 4 shows the counting results for aggregatedplatelets in the presence of increasing doses of antibody CD9 (clone6B1).

Example 6

[0149] Platelet Aggregation with the CD9 Antibody in the Presence ofBacteria

[0150] Method:

[0151] a cryobead of Staphylococcus epidermidis was introduced into atube of tryptone soy broth and incubated at 37° C. for 18-24 hours;

[0152] 400 μl of platelet concentrate placed in tubes was added to 50 μlof the Staphylococcus epidermidis suspension and 50 μl of CD9;

[0153] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0154] 500 μl of PBS buffer was added;

[0155] filtration was performed on a filter with a porosity of 5 μm;

[0156] dilutions were performed in series: four dilutions {fraction(1/10)};

[0157] filtration of 100 μl of the sample was performed on a CB04membrane;

[0158] labeling was performed with an esterase; and

[0159] detection was performed.

[0160] Table 4 below illustrates platelet aggregation with CD9 in thepresence of bacteria. TABLE 4 Bacteria CD9 Result % recovery Staph. PC10 — 46 93 epidermidis 44 Staph. PC 10 +agitation 43 epidermidis 48 E.coli PC 10 218 64 243 E. coli PC 10 CD9 + agitation 166 148

[0161] These results show that the Staphylococcus epidermidis bacteriawere recovered in a pronounced manner. For E. coli, the count wasreduced by 36%.

[0162] I.2 Agglutination of Red Cells

[0163] Lectins are glycoproteins of nonimmune origin that agglutinatecells and/or precipitate carbohydrate complexes. These molecules readilybond with specific carbohydrates.

[0164] Two lectins were used: Phaseolus vulgaris PHA-E and Vicia sativa.

Example 1

[0165] Agglutination of Red Cells with Lectins

[0166]Phaseolus vulgaris PHA-E lectin (Sigma) was used at aconcentration of 2 mg/ml in PBS. Vicia sativa lectin (Sigma) was used atthe concentration of 1 mg/ml. A quick evaluation of the two lectinsrevealed that there is no red cell agglutination with Vicia sativa. Onthe other hand, Phaseolus vulgaris induced a rapid and effectiveagglutination.

[0167] Method:

[0168] 400 μl of red cells were placed in tubes to which were added 50μl of PBS and 50 μl of Phaseolus vulgaris;

[0169] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0170] 500 μl of PBS buffer was added;

[0171] filtration was performed on a filter with a porosity of 5 μm;

[0172] dilutions were performed in series: four {fraction (1/10)}dilutions;

[0173] labeling was performed with an anti-glycophorine-PE antibody; and

[0174] detection was performed.

[0175]FIG. 6 shows agglutination of the red cells obtained in thepresence of Phaseolus vulgaris. Phaseolus vulgaris was used in this testat a concentration of 200 μg/ml. We saw a reproducible decrease of twologs in the concentration of red cells in the presence of Phaseolusvulgaris.

Example 2.

[0176] Agglutination of Red Cells in the Presence of Bacteria

[0177] The preliminary tests showed that there is no interaction betweenthe Phaseolus lectin and the bacteria.

[0178] Method:

[0179] a cryobead of E. coli was introduced into a tube of 9 ml oftryptone soy broth and incubated at 37° C. for 18-24 hours;

[0180] 400 μl of PBS, 50 μl of E. coli (pure culture) and 50 μl ofPhaseolus vulgaris was mixed in the tubes;

[0181] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0182] 500 μl of PBS buffer was added;

[0183] filtration was performed on a 5-μm filter;

[0184] dilutions were performed in series: four dilutions {fraction(1/10)};

[0185] filtration was performed on 100 μl of the sample on a CB04membrane;

[0186] labeling was performed with an esterase; and

[0187] detection was performed.

[0188] The results obtained using a Phaseolus vulgaris concentration of200 μg/ml are shown in Table 5 below. TABLE 5 Lectin Bacteria Red Cells(concentration) Count % recovery E. coli RBC 9 662 NCTC 9001 604 E. coliRBC 9 Phaseolus 200 ug/ml 544 91 NCTC 9001 579

[0189] These results show that 91% of the bacteria were detected afterthe agglutination step and that after this agglutination step, the redcell concentration was reduced by two logs while the strain E. coli wasstill recovered in a pronounced manner.

[0190] II. Concentration of Pathogenic Microbes by Means of a Lysis Step

[0191] We evaluated the different selective lysis techniques enablingelimination of blood cells without affecting the concentration of thebacteria possibly present in the samples to be analyzed.

[0192] Following several preliminary studies, we observed that certainbacteria were resistant in the presence of detergents such as TritonX100 and determined the concentration of detergents with which thebacteria recovery percentage is desired.

Example 1

[0193] Effect of the Formulation of the Lysis Solution on Pure BacterialCultures

[0194] Method:

[0195] the strains were preserved in a tube of 9 ml of tryptone soybroth and incubated at 37° C. for 18-24 hours;

[0196] dilutions in series ({fraction (1/10)}) were performed in the PBSbuffer up to 10-5;

[0197] one milliliter of the last dilution was treated with 9 ml of thelysis solution 0.01% (weight/volume) of saponin, 0.1% (weight/volume) ofTween and 0.001% (weight/volume) of Triton X 100 for 15 minutes;

[0198] 100 μl of the sample was filtered on a CB04 membrane;

[0199] labeling was performed with an esterase; and

[0200] detection was performed.

[0201] The results of these different tests are illustrated in Table 6below in which are expressed the different bacteria recovery percentagesobtained. TABLE 6 Strain Control Lysed sample % recovery E. coli 132 15398 145 119 Bacillus cereus 465 61 11 572 54 E. coli 52 44 83 54 44Staph. epidermidis 2952 3024 101 3069 3020 E. aerogenes 841 880 110 802925 Ps. aeruginosa 261 82 31 215 66 Staph. aureus 105 126 125 94 122 P.mirabilis 729 1129 139 906 1151 S. typhimurium 608 820 133 600 787Serratia marcescens 1848 1826 102 1775 1860 C. amycolatum 1103 1512 1561167 2035 K. pneumoniae 72 79 99 82 73 P. fluorescens 4039 4873 125 39515105 Streptococcus bovis 3074 1848 64 2939 1978 Y. enterocolitica 10,2189888 99 10,321 10,526

[0202] These results are illustrated in FIG. 7.

[0203] Most of the strain are not affected by the lysis solution. Therecovery percentages are consistent predefined predictions, between 85and 115%, except for Ps. aeruginosa and Bacillus cereus.

Example 2a

[0204] Effect of the Formulation of the Lysis Solution on the BacteriaSeeded in the Platelets

[0205] Two strains were adapted to the growth in the plateletconcentrates to simulate a contamination.

[0206] Method:

[0207] the strains Bacillus cereus and Staph. aureus were seeded inplatelets in 50-ml tubes using a concentration of 10⁶ cells in 20 ml ofplatelets;

[0208] the 50-ml tubes were kept in the platelet incubator at 22° C. forseveral days;

[0209] one milliliter of seeded platelets was diluted with 9 ml of thelysis solution;

[0210] lysis was performed for 15 minutes at ambient temperature;

[0211] 100 μl of the sample was filtered on a CB047 membrane;

[0212] the bacteria were labeled with a stearic substrate; and

[0213] the membrane was scanned with the analyzer.

[0214] The bacteria count results after lysis are illustrated in Table 7below. TABLE 7 Control Lysed Bacillus cereus bacteria/ml  16  14  24  272.0E+06 2.1E+06 Staph. aureus bacteria/ml 1966 1885 2046 1901 2.0E+081.9E+08

[0215] The bacteria were seeded at an initial concentration of 5·10³cells/ml and detected several days later at concentrations on the orderof 10⁶ to 10⁹ cells/ml. It can be seen from these experiments that thebacteria that developed in the platelets were not affected by the lysissolution.

Example 2b

[0216] Effect of the Formulation of the Lysis Solution on the PureBacteria Cultures

[0217] Method:

[0218] the strains were preserved in a tube of 9 ml of tryptone soybroth and incubated at 37° C. for 18-24 hours;

[0219] after determination of the number of bacteria by esteraselabeling, 3000 bacteria were inoculated in 3 mL of PBS;

[0220] the 3 mL were treated with the lysis solution (NOG 0.25% to 2%)for 20 minutes;

[0221] the totality of the sample was filtered on a CB04 membrane; and

[0222] counting was performed in solid phase cytometry.

[0223] The results obtained are illustrated in FIG. 11.

Example 3

[0224] Effect of the Formulation of the Lysis Composition on the RedCells

[0225] Method:

[0226] one milliliter of red cells was diluted in 9 ml of lysis solutionor 9 ml of PBS (control);

[0227] lysis was performed for 15 minutes at ambient temperature; and

[0228] the lysed or non-lysed samples were analyzed using a cellcounter.

[0229]FIG. 9 shows the results obtained on the lysed red cellpreparation compared to the non-lysed red-cell preparation.

Example 4

[0230] Effect of the Formulation of the Lysis Composition on thePlatelets

[0231] Reproducibility of the efficacy of the lysis solution was tested.Different platelet samples were lysed and analyzed.

[0232] Method:

[0233] one milliliter of platelets was diluted in 9 ml of lysissolution;

[0234] lysis was performed for 15 minutes at ambient temperature;

[0235] dilutions in series ({fraction (1/10)}) were created in the PBSbuffer up to 10⁻⁵;

[0236] 100 μl of the sample was filtered on a CB04 membrane;

[0237] the microorganisms were labeled with an esterase substrate; and

[0238] the membrane was scanned with the analyzer.

[0239]FIG. 8 illustrates the lysis results obtained with differentplatelet samples.

[0240] III. Concentration of Pathogenic Microbes by Two Aggregation andLysis Steps

[0241] Preparation of the sample by concentration of the pathogenicmicrobes in two steps comprised:

[0242] 1) specific aggregation or agglutination of the cells of theblood product, and

[0243] 2) specific lysis of the cells of the blood product.

Example 1

[0244] Specific Separation of the Platelets

[0245] Method:

[0246] A cryobead of E. coli was introduced in a tube of 9 ml oftryptone soy broth and incubated at 37° C. for 18-24 hours. The firstaggregation step was performed as follows:

[0247] 1 ml of platelet concentrate, 50 μl of E. coli (pure culture) and100 μl of CD9 was mixed in the tubes;

[0248] the tubes were agitated manually for 5 minutes at ambienttemperature.

[0249] The second lysis step was then performed:

[0250] 900 μl of lysis buffer was added to 100 μl of filtrate afteraggregation;

[0251] the mixture was maintained for 15 minutes at ambient temperature;

[0252] dilutions were performed in series: four dilutions {fraction(1/10)};

[0253] 100 μl of the resultant sample was filtered on a CB04 membrane;

[0254] the bacteria and platelets were labeled with an esterasesubstrate; and

[0255] the bacteria and platelets were counted with the analyzer.

[0256] The results obtained in performing the method for theconcentration of bacteria in two steps are illustrated in Table 8 below.TABLE 8 Platelet % bacterial Bacteria concentrate (PC) Aggregation agentBacteria count recovery E. coli PC 337 NCTC 9001 313 E. coli PC lysis302 86% NCTC 9001 256 E. coli PC CD9 266 66% NCTC 9001 161 E. coli PCCD9 lysis 157 45% NCTC 9001 138

Example 2

[0257] Specific Separation of the Red Cells

[0258] Method:

[0259] a cryobead of E. coli was introduced into a tube of 9 ml oftryptone soy broth and incubated at 37° for 18-24 hours.

[0260] The agglutination step was performed as follows:

[0261] 1 ml of red cells, 50 μl of E. coli (pure culture) and 125 μl oflectin was mixed in the tubes;

[0262] the tubes were agitated manually for 5 minutes at ambienttemperature;

[0263] filtration was performed on a filter with a porosity of 5 μm.

[0264] The second lysis step was then performed:

[0265] 900 μl of lysis buffer was added to 100 μl of the filtrate afteragglutination;

[0266] the mixture was maintained for 15 minutes at ambient temperature;

[0267] dilutions were performed in series: four dilutions {fraction(1/10)};

[0268] 100 μl of the resultant sample was filtered on a CB04 membrane;

[0269] the bacteria were labeled with an esterase substrate and of thered cells with an anti-glycophorin-PE antibody; and

[0270] the bacteria and red cells were counted with the analyzer.

[0271] Table 9 below shows the results obtained with the counting of thebacteria performed after the step of agglutination of the red cells withlectin followed by the lysis step. TABLE 9 Bacteria Bacteria recoveryRed cell counts (%) counts E. coli Red cells 10 441 3582 NCTC 9001 4494188 E. coli Red cells 10 lectin lysis 261 68 161 NCTC 9001 348 180

[0272] The red cell concentration was reduced by 1.5 log afteragglutination. 68% of the bacteria were recovered after these two steps.

[0273] IV. Marker Agents

Example 1

[0274] Esterase substrate ChemChrome V6

[0275] Marker solutions can be prepared from an esterase substrate andused in the detection method of the invention according to the followingprotocol:

[0276] a) Preparation

[0277] 10 μl of esterase substrate ChemChrome V6 per milliliter ofChemSol B16 buffer (500 μl of marker solution per membrane); and

[0278] this solution was stored at 4° C. shielded from light for amaximum of 4 hours.

[0279] b) Use

[0280] introduce a labeling buffer into a 33-mm diameter Petri dish;

[0281] distribute 500 μl of the marker solution on the buffer; and

[0282] place the CB04 membrane on the filtration gradient. Filter 100 μlof the sample to be analyzed;

[0283] place the membrane on the buffer; and

[0284] incubate for 15 minutes at 37° C.

Example 2

[0285] Labeled Antibody

[0286] Thus, a marker solution comprising a labeled antibody can be usedaccording to the following protocol:

[0287] collect 90 μl of a dilution of the sample;

[0288] add 10 μl of the anti-glycophorin-PE antibody;

[0289] vortex and incubate for 15 minutes at ambient temperatureshielded from the light;

[0290] add 900 μl of PBS buffer;

[0291] place the CB04 membrane in the filtration gradient;

[0292] filter under vacuum 100 μl of the solution to be analyzed; and

[0293] analyze the membrane in the analyzer, reversing the primary andtertiary cables of the analyzer.

Example 3

[0294] Value of the Addition of a Bacteria Permeabilization Agent toImprove the Penetration of the Marker

[0295] Method:

[0296] the strains were preserved in a tube of 9 ml of tryptone soybroth and incubated at 37° C. for 18-24 hours;

[0297] after determination of the bacteria count by esterase labeling,3000 bacteria were inoculated in 3 mL of platelet concentrate;

[0298] the 3 mL were treated with 1 ml of the aggregation solution (CD9:clone 6B1: 30 μg/ml, Picogreen {fraction (1/2000)}, PEI 40 μg/mL at 80μg/mL) for 40 minutes;

[0299] the sample was filtered through a filter with a porosity of 5 μm;

[0300] the sample was incubated in the lysis solution (chlorhexidine5·10−³%, NOG 0.5%, nisin 0.2 μg/ml, EDTA 5 mM) for 20 minutes;

[0301] the totality of the sample was filtered on a CB04 membrane; and

[0302] counting was performed by means of a cytometer analyzer in solidphase.

[0303] V. Conclusions

[0304] The technical options for achieving the excellent conditions forthe preparation of pathogenic microbes were defined by theseexperiments.

[0305] With regard to the platelet concentrates, these technical optionscomprise:

[0306] 1) an aggregation step with, e.g., a platelet activator antibodysuch as CD9;

[0307] 2) a cell lysis step with a combination of detergents such assaponin, Tween 20 and Triton X 100.

[0308] With regard to the red cell concentrates:

[0309] 1) an aggregation step with a lectin such as, e.g., Phaseolusvulgaris;

[0310] 2) a cell lysis step with a combination of detergents such assaponin, Tween 20 and Triton X 100.

[0311] Labeling and permeabilization of the pathogenic microbes can beperformed as desired during the aggregation step, the lysis step ordirectly on the concentrated microbes on the last filter beforeanalysis.

1. A method for detecting contaminating microbes possibly present in ablood product comprising blood cells comprising: a) subjecting a sampleof the blood product to an aggregation treatment of the blood cells, b)substantially eliminating aggregates formed in step (a) by passage ofthe sample over a first filter allowing passage of contaminatingmicrobes, but not cell aggregates, c) selectively lysing residual cellsof the filtrate obtained in step (b), d) recovering the contaminatingmicrobes by passage of the lysate from step (c) over a second filterallowing passage of cellular debris, e) adding a marker agent of thecontaminating microbes either during step (a) or step (c), and f)analyzing material on the second filter to detect labeled contaminatingmicrobes possibly retained by the second filter.
 2. The method accordingto claim 1, further comprising addition of a permeabilization agent ofthe contaminating microbes in at least one of the steps (a), (c) or (e).3. The method according to claim 2, wherein the permeabilization agentis selected from the group consisting of polyethylene imine,chlorhexidine diacetate, chlorhexidine digluconate, ethylene diaminetetraacetate acid (EDTA) alone or in combination with nisin, a detergentand mixtures thereof.
 4. The method according to claim 3, wherein thedetergent is selected from the group consisting of N-octylβ-D-glucopyranoside, SDS, Tween, triton, Brij and mixtures thereof. 5.The method according to claim 1, wherein the marker agent is a markersolution selected from among the group consisting of an esterasesubstrate, a labeled antibody and a marker of nucleic acids.
 6. Themethod according to claim 1, wherein the marker agent comprises afluorescent marker or an agent coupled to a fluorochrome or an enzymeenabling degradation of a substrate thereby made fluorescent.
 7. Themethod according to claim 6, wherein fluorescence is detected by anexcitation laser.
 8. The method according to claim 1, wherein the bloodcells of the blood product are platelets or red cells or a mixturethereof.
 9. The method according to claim 1, wherein the blood productcomprises platelets and step (a) comprises bringing the sample intocontact with an aggregation composition comprising at least oneaggregation agent selected from the group consisting of 1) a specificantibody of a platelet antigen, 2) a strong agonist of plateletactivation selected from the group consisting of thrombin, TRAP(thrombin receptor activating peptide), trypsin, collagen, thromboxaneA2, PAF (platelet activating factor), ionophore A23187, immune complexesand complement factors, and 3) a weak agonist of platelet activationselected from the group consisting of ADP, adrenalin, arachidonic acid,Von Willebrand factor, serotonin and epinephrine.
 10. The methodaccording to claim 9, wherein concentration of the antibody is betweenabout 0.5 μg/ml and about 50 μg/ml.
 11. The method according to claim 9,wherein concentration of strong agonist is between: about 0.5 IU/ml andabout 100 IU/ml for thrombin; about 5 μM and about 200 μM for TRAP;about 1 nM and about 500 nM for trypsin; about 0.05 μg/ml and about 50μg/ml for collagen; about 0.01 μg/ml and about 5 μg/ml for thromboxaneA2; about 0.005 mg/ml and about 1 mg/ml for PAF; or about 0.1 μM andabout 100 μM for ionophore A23187.
 12. The method according to claim 9,wherein concentration of the weak agonist is between: about 0.5 μM andabout 100 μM for ADP, adrenalin or epinephrine; about 0.001 mM and about10 mM for arachidonic acid; about 0.001 mg/ml and about 1 mg/ml for VonWillebrand factor; or about 0.05 μ and about 100 μM for serotonin. 13.The method according to claim 9, wherein the antibody is selected fromthe group consisting of an anti-CD, anti-CD32, anti-PTA1, anti-D42,anti-GpIIb/IIIa and anti-GpIV antibody.
 14. The method according toclaim 1, wherein the blood product comprises red cells and step (a)comprises bringing the sample into contact with an agglutinationcomposition comprising at least one agglutination agent selected fromthe group consisting of lectins, polyethylene imine,polyvinylpyrrolidone (PVP), gelatins, dextrans and polyethylene glycols(PEG).
 15. The method according to claim 14, wherein the lectins haveerythroagglutinin activity.
 16. The method according to claim 14,wherein the lectins are selected from the group consisting of Phaseolusvulgaris, Vicia sativa, Vicia faba and Erythrina corallodendron.
 17. Themethod according to claim 16, wherein concentration of Phaseolusvulgaris lectin is between about 10 μg/ml and about 200 μg/ml.
 18. Themethod according to claim 14, wherein concentration of polyethyleneimine is between about 0.1% (weight/volume) and about 40%(weight/volume).
 19. The method according to claim 14, wherein thepolyvinylpyrrolidone (PVP) is selected from the group consisting ofPVP-40 and PVP-360 at a concentration between about 0.1% (weight/volume)and about 40% (weight/volume).
 20. The method according to claim 14,wherein the gelatin is at a concentration between about 0.5%(weight/volume) and about 40% (weight volume).
 21. The method accordingto claim 14, wherein dextran is selected from the group consisting ofDextran 70, Dextran 100 and Dextran 500 at a concentration between about0.1% (weight/volume) and about 40% (weight/volume).
 22. The methodaccording to claim 14, wherein the polyethylene glycol is selected fromthe group consisting of PEG8, PEG17 and PEG35 at a concentration betweenabout 0.05% (weight/volume) and about 40% (weight/volume).
 23. Themethod according to claim 1, wherein step (c) is performed with a lysissolution comprising one or more detergents selected from the groupconsisting of saponin, SDS, Tween 20, Triton X100, Brij 96, Polidocanol,N-octyl β-D-glucopyranoside and sodium carbonate.
 24. The methodaccording to claim 1, wherein the contaminating microbes are aerobic oranaerobic bacteria, molds, yeasts, or live and/or dead bacterial spores.25. The method according to claim 1, wherein the size of pores of thefirst filter are between about 2 μm and about 20 μm.
 26. The methodaccording to claim 1, wherein the size of pores of the second filter arebetween about 0.2 μm and about 2 μm.
 27. The method according to claim1, wherein the contaminating microbes are aerobic or anaerobic bacteria,molds, yeasts, or live and/or dead bacterial spores, and the size ofpores of the first filter are between about 2 μm and about 20 μm, andthe size of pores of the second filter are between about 0.2 μm andabout 2 μm.
 28. The method according to claim 1, wherein step (f) isperformed in an enclosed device.
 29. A device for concentratingcontaminating microbes possibly present in a blood product comprisingblood cells comprising: a first watertight, sterile tank containing atleast one blood cell aggregation agent and, optionally, at least oneagent for labeling pathogenic microbes; a second watertight, steriletank containing at least one lysis agent for blood cells and,optionally, at least one agent for labeling pathogenic microbes; a firstfilter located between the first and second tanks and capable ofretaining aggregates formed in said first tank; a second filter locateddownstream of the second tank and capable of retaining possiblecontaminating pathogenic microbes; and a watertight, sterile connectorplaced between the first tank and the first filter, between the firstfilter and the second tank, and between the second tank and the secondfilter.
 30. The device according to claim 29, further comprising awatertight, sterile connector to connect a bag containing the bloodproduct to the first sterile tank.
 31. The device according to claim 30,wherein the watertight, sterile connection connecting the bag containingthe blood product to the first sterile tank has a reverse lock valve.32. The device according to claim 29, further comprising means forsampling a determined volume of the blood product directly from astorage bag of the product into the first tank.
 33. The device accordingto claim 30, wherein the first sterile tank is fitted with a samplesuctioning system.
 34. The device according to claim 33, wherein thesuctioning system is a piston.
 35. The device according to claim 29,wherein the second filter is enclosed in a membrane support having twoparts that can be separated for removing the filter.
 36. The deviceaccording to claim 29, which is enclosed and sterile.